This invention relates to a method for introducing functional nucleic acids into cells using a bacterial delivery system. A bacterial vector capable of delivering functional nucleic acids to cells can be produced by introducing a bacterial plasmid containing promoters and other sequences containing instructions recognized by eukaryotic cells into bacteria capable of invading cells, being taken up by cells, or interacting with cells in such a way as to have the nucleic acids reach the eukaryotic cell cytoplasm. The nucleic acids delivered to the cell in this way can direct the eukaryotic cell to produce antigens or other functional molecules.
These unique bacterial delivery systems therefore can be used as vaccines to prevent or treat infectious diseases and cancer, down regulate the immune system in the case of tissue rejection in transplantation, prevent or treat autoimmune diseases and other diseases related to dysregulation of the immune system. In addition, the bacterial delivery systems can be used for gene therapy or gene replacement for treatment or amelioration of disease such as hereditary genetic diseases, cancers and virus infections.
Direct DNA-mediated immunization is another approach to the introduction of functional nucleic acids and vaccine development. Highly purified bacterial plasmid DNAs expressing desired proteins under the control of viral promoters have been injected primarily into muscle or skin by traditional needle and syringe or by other more exotic methods such as biolistic transfection with DNA-coated gold microparticles (for review see Donnelly, J. J. et al. J. Immunol Methods (1994) 176: 145) (All documents cited herein supra or infra are hereby incorporated by reference). Investigators using this technology have been able to elicit neutralizing antibodies, cytotoxic T lymphocytes and protection against challenge in several animal models of infection ranging from influenza to malaria. The use of bacteria as a delivery system as described in this invention is a unique method of delivering DNA to mammalian cells and has the potential to provide a simple, inexpensive way of extending DNA immunization to the local immune system and beyond through oral and other mucosal routes of immunization.
Previously, live bacteria have been utilized as vaccines in order to protect against subsequent infection. Attenuated or less virulent Shigella, Salmonella, Listeria, and other bacteria have been given orally to immunize against subsequent infection with more virulent forms of these bacteria. Likewise, attenuated bacterial and mycobacterial organisms such as Bacille Calmette-Guerin (BCG) have been administered parenterally to protect against related organisms such as M. tuberculosis. Genes from bacteria, viruses and parasites have been cloned into a variety of bacteria and mycobacteria for the purpose of directing the bacteria to express the foreign antigen or impart on the bacteria certain desired properties for use as a live vaccine. Examples include cloning the invasion genes of Shigella into the normally non-invasive E. coli rendering the E. coli invasive and therefore more suitable for use as a vaccine strain, or cloning of P. falciparum malaria genes into Salmonella typhimurium which subsequently express these malaria proteins and, following oral administration of the bacteria, induce specific cytotoxic T cell immunity and protection in mice against malaria challenge (Sadoff et al. Science (1988) 240 :336-338; Aggrawal et al. J. Exp. Med. (1990) 172:1083-1090). All of these bacterial delivery systems require the bacteria itself to produce the antigen or functional molecule and are dependent on a bacterium which is sufficiently attenuated to be safe for use in humans, but still able to induce a protective response. The bacterial delivery system of the present invention is designed to deliver functional nucleic acids which are then transcribed and translated as directed by the eukaryotic machinery to produce foreign antigens or functional molecules. In this case, antigen toxicity which is often seen when using live attenuated bacterial carriers expressing foreign proteins/peptides is eliminated because the expression occurs within the mammalian via its own machinery. This also will allow for any secondary modifications required of the protein; thus, permitting the protein to take a more charactistic/natural form for processing and presentation to the immune system or to direct a cellular function. In addition, if desired, it can be used to deliver prokaryotically produced antigens and functional molecules.
This invention can be applied to any desired bacteria. We chose Shigella as an example of a bacterial delivery system because of its ability to invade cells, escape from the endocytic vacuole, and enter into the cytoplasm of eukaryotic cells. These properties are not required of a bacteria chosen for application of the present invention, but simplified the experimental system. Shigellae are enteric pathogens that invade the human colonic epithelium and multiply intracellularly, causing bacillary dysentery. Bacillary dysentery is caused by all members of the genus Shigella (S. boydii, S. dysenteriae, S. flexneri, and S. sonnei). Shigellosis is prevalent in developing countries, but is also found in industrialized nations, especially in institutional settings. It has been estimated that Shigellosis is the cause of half a million deaths a year, mostly among children, making the development of a safe and effective Shigella vaccine important (Stole, B. J. et al. J. Infect. Dis. (1982) 146: 177). To cause dysentery, Shigella strains must be able to recognize, invade and multiply within epithelial cells of the colon (LaBrec, E. H. et al. J. Bacteriol. (1964) 88: 1503). Both the bacteria and host cell play a role in the invasive process wherein the host cell actively engulfs the bacteria which in turn escapes from the phagosome by a bacteria-mediated digestion of the phagosomal membrane (Sansonetti, P. J. et al. Infect. Immun. (1981) 34: 75). Once in the cell, bacterial multiplication occurs resulting in host cell necrosis.
In this invention is described a method for delivering DNA to cells. The method of the present invention includes the introdution of the desired DNA into bacteria, allowing the bacteria to infect or enter cells, and then lysing the bacteria inside the cells such that the desired DNA is released. Even though a specific bacteria is described herein and is shown to deliver nucleic acids to eukaryotic cells, this invention is applicable to all bacteria and mycobacteria. Plasmids introduced into other cells such as plant cells may also render these cells capable of delivering nucleic acids.
Specifically, the method of the present invention uses an attenuated bacterial strain which is sufficiently attenuated to not cause disease, while still maintaining the ability to enter mammalian cells. The attenuated strain used in the method of the present invention does not carry genetic mutations specifically designed to lyse the carrier and mediate delivery of plasmid DNA. Since the bacterial strain is not engineered to lyse, delivery of the DNA in the cell is mediated by alternative methods, such as, for example, the use of antimicrobial agents. The Shigella flexneri strain used as a model in the present invention, SC602, carries mutations in the icsA gene required for intracellular spread and aerobactin (iuc:: iut) and was described in Barzu et al., Infection and Immunity 64: 1190-1194 (1996). Once the attenuated bacteria containing the DNA to be delivered is inside the cell, antimicrobials are introduced which eliminate the bacteria and allow the release of intact DNA into the cell.
The advantage of the method of the present invention is that any bacterial strain can be used; so long as the strain meets the following criteria: attenuated for use in humans or other animals, invasive, and exits or breaks down the endocytic or phagocytic vacuole in such a way as to allow the release of functional DNA. Therefore, one can take advantage of the higher level of invasiveness afforded other attenuated bacterial strains thereby increasing the number of mammalian cells infected, and thus the number of mammalian cells expressing the foreign protein or peptide once the bacteria is lysed inside the cell.
Therefore, it is one object of the invention to provide a delivery vehicle for the delivery of DNA to cells. The DNA encoding desired gene(s) or antigen(s) along with control sequences can be introduced into attenuated bacteria, and the recombinant attenuated strain allowed to enter mammalian cells afterwhich antimicrobials able to lyse the bacteria are introduced such that the bacteria is lysed and DNA delivery is mediated. Such a delivery vehicle could be used for oral and other mucosal immunization and gene therapy strategies.
It is another object of the present invention to provide a Shigella delivery vehicle for the delivery of DNA to mucosal surfaces. The DNA encoding desired gene(s) or antigen(s) can be introduced into attenuated Shigella, the recombinant attenuated Shigella strain allowed to enter mammalian cells, afterwhich, antimicrobials able to lyse the Shigella are introduced such that the Shigella is lysed and DNA delivery is mediated. Such a delivery vehicle could be used for oral and other mucosal immunization and gene therapy strategies.
It is still another object of the present invention to provide a method for the delivery of heterologous foreign antigens expressed by attenuated bacteria, more specifically Shigella, for the purpose of inducing in an individual an immune response against the foreign antigen or for treatment of a disease wherein said foreign antigen is missing or found in reduced amount.
It is further another object of the invention to provide a delivery vehicle for delivery of functional DNA and antigens to cells in vitro for use of those cells in, for example, transplantation and gene therapy. The DNA encoding desired gene(s) or antigen(s) can be introduced into attenuated bacteria, and the recombinant attenuated strain allowed to enter cells in vitro afterwhich, antimicrobials able to lyse the bacteria are introduced such that the bacteria is lysed and DNA delivery is mediated.
It is yet another object of the invention to provide a general method for introducing functional DNA into cells using bacterial delivery systems. The desired functional DNA is introduced into an attenuated bacteria able to enter or infect cells afterwhich, an antimicrobial is used to lyse the bacteria inside the cell such that the DNA is released in the cell. This method can be used for inducing protective immunity as a vaccine, for preventing and treating tumors, for the therapy and treatment of autoimmune disorders, for the treatment of conditions related to dysfunction of the immune system, for transplantation, for gene replacement, and gene therapy.